In recent years, an increased interest in the phyto-chemistry of the Annonaceae has been sparked by the bioactivity-directed isolation of the antileukemic Annonaceous acetogenin, uvaricin, from Uvaria acuminata. Acetogenins are C.sub.35 -C.sub.39 compounds and typically contain two long hydrocarbon chains, one of which connects a terminal 2,4-disubstituted-.gamma.-lactone to a variable number of tetrahydrofuran (THF) rings. The hydrocarbon chains contain a number of oxygenated moieties which can be hydroxyls, acetoxyls and/or ketones.
All acetogenins found to date contain multiple stereocentres, the elucidation of which often presents daunting stereochemical problems. Because of their waxy nature, the acetogenins do not produce crystals suitable for X-ray crystallographic analysis. Relative stereochemistries of ring junctions have typically been determined by comparison of natural compounds with synthetic model compounds and such methods have proven to be invaluable with the acetogenins. Recently, the absolute stereochemistries of the carbinol centers of acetogenins have been determined with the help of synthetic model compounds and high field nuclear magnetic resonance (NMR) analysis of their methoxyfluoromethylphenylacetic acid (MPTA) esters (Mosher esters).
Most Annonaceous acetogenins are potently bioactive, but the mode of action of these compounds was unknown until Londerhausen et al. concluded in Pesticide Science, 33, 427-438 (1991), that they act to inhibit complex I of mitochondrial oxidative phosphorylation with an activity several times that of rotenone.
Annona squamosa, the custard apple, is a fruit tree, native to the new world and naturalized throughout the tropics. Research on the seeds of this plant has yielded 27 different acetogenins. By contrast, only four have been reported from the bark, and these are bullatacin, (3), 4-cis- and 2,4-trans-bullatacinones, and squamone. Through further work with the bark, each of these four known compounds have now been reisolated. Additional compounds have been isolated from this species, including asimicin, which is new to the bark of this species, molvizarin (2), which is new to the species, and squamotacin (1), which is a new acetogenin. The structure of squamotacin (1), molvizarin (2) and bullatacin (3) are shown below. ##STR1##
Molvizarin and squamotacin were isolated from Annona squamosa using column chromatography followed by HPLC. The active fractions containing these compounds were identified based on their bioactivity as revealed by the brine shrimp lethality test (BST), detailed in Example 2. Structure elucidation of the isolated compounds was carried out using NMR and MS analyses, the relative stereochemistry of squamotacin was suggested based on the close agreement with the NMR signals of bullatacin whose absolute stereochemistry is now known through Mosher ester analyses.
The present invention is directed to the isolation of two natural cytotoxic acetogenin compounds, squamotacin and molvizarin, and the preparation of chemotherapeutic compositions comprising those compounds for use in treating patients having tumors.